pone-0104293-g001: CDX2 is alternatively spliced.(A) Sequence analysis of CDX2 mRNA isolated from normal colonic epithelial cells revealed wild type mRNA and a variant transcript (CDX2/AS) lacking the bases CAGG. (B) Alternative splicing of CDX2 occurs via utilization of the non-canonical splice donor sequence GC located at the 3′ region of exon 2. (C) The alternatively induced frameshift results in deletion of the carboxy-terminal 85 bases in the wild type protein that are replaced by a novel 45 domain enriched in serine and arginine residues. (D) Western blot analysis of whole cell lysates from a normal colonic epithelium and from the HT29 colorectal cancer cell line blotted with anti-CDX2/AS revealed a band at 38 kD. (E) Western blot analysis of whole cell lysates from normal adjacent colonic epithelium (N) and colon adenocarcinomas (T) as well as whole cell lysates from various colon cancer cell lines blotted with antibody CDX2ACT that recognizes the amino-terminal domain of CDX2 and CDX2/AS revealed two bands at 42 and 38 kD representing CDX2 and CDX2 kD representing CDX2 and CDX2/AS, respectively.

Mentions:
We sequenced full-length CDX2 cDNA clones generated by RT-PCR using RNA isolated from normal human colonic mucosa and identified two transcripts, wild type CDX2 and a variant transcript (CDX2/AS) lacking the four bases CAGG (Fig. 1A) (Genbank ID: KJ081251). Sequence analysis of the genomic CDX2 region corresponding to the four base pair deletion revealed an intact gene (data not shown). Given these findings, we attributed the generation of CDX2/AS to alternative splicing in which the non-canonical 5′ splice donor sequence, GC, located at the distal segment of exon 2 is utilized rather than the wild type GU donor sequence of intron 2 (Fig. 1B). The alternative splicing-induced frameshift occurs in the distal region of the homeobox binding domain and encodes a protein in which the 85 carboxyl terminal residues in the wild type protein are replaced with a novel 45 residue domain enriched in serine and arginine amino acids (Fig. 1C). Western blot analysis of whole cell lysates from normal colonic mucosa and from the HT29 colorectal cancer cell line using polyclonal antisera to the unique carboxy-terminal domain of CDX2/AS revealed a 38 kD band representing the alternatively spliced protein (Fig. 1D). Using an antibody recognizing the common amino-terminal activation domain, proteins at 42 kD (CDX2) and 38 kD (CDX2/AS) were identified in whole cell lysates from human colonic tumors, normal adjacent colonic mucosa, and several colon cancer cell lines (Fig. 1E).

pone-0104293-g001: CDX2 is alternatively spliced.(A) Sequence analysis of CDX2 mRNA isolated from normal colonic epithelial cells revealed wild type mRNA and a variant transcript (CDX2/AS) lacking the bases CAGG. (B) Alternative splicing of CDX2 occurs via utilization of the non-canonical splice donor sequence GC located at the 3′ region of exon 2. (C) The alternatively induced frameshift results in deletion of the carboxy-terminal 85 bases in the wild type protein that are replaced by a novel 45 domain enriched in serine and arginine residues. (D) Western blot analysis of whole cell lysates from a normal colonic epithelium and from the HT29 colorectal cancer cell line blotted with anti-CDX2/AS revealed a band at 38 kD. (E) Western blot analysis of whole cell lysates from normal adjacent colonic epithelium (N) and colon adenocarcinomas (T) as well as whole cell lysates from various colon cancer cell lines blotted with antibody CDX2ACT that recognizes the amino-terminal domain of CDX2 and CDX2/AS revealed two bands at 42 and 38 kD representing CDX2 and CDX2 kD representing CDX2 and CDX2/AS, respectively.

Mentions:
We sequenced full-length CDX2 cDNA clones generated by RT-PCR using RNA isolated from normal human colonic mucosa and identified two transcripts, wild type CDX2 and a variant transcript (CDX2/AS) lacking the four bases CAGG (Fig. 1A) (Genbank ID: KJ081251). Sequence analysis of the genomic CDX2 region corresponding to the four base pair deletion revealed an intact gene (data not shown). Given these findings, we attributed the generation of CDX2/AS to alternative splicing in which the non-canonical 5′ splice donor sequence, GC, located at the distal segment of exon 2 is utilized rather than the wild type GU donor sequence of intron 2 (Fig. 1B). The alternative splicing-induced frameshift occurs in the distal region of the homeobox binding domain and encodes a protein in which the 85 carboxyl terminal residues in the wild type protein are replaced with a novel 45 residue domain enriched in serine and arginine amino acids (Fig. 1C). Western blot analysis of whole cell lysates from normal colonic mucosa and from the HT29 colorectal cancer cell line using polyclonal antisera to the unique carboxy-terminal domain of CDX2/AS revealed a 38 kD band representing the alternatively spliced protein (Fig. 1D). Using an antibody recognizing the common amino-terminal activation domain, proteins at 42 kD (CDX2) and 38 kD (CDX2/AS) were identified in whole cell lysates from human colonic tumors, normal adjacent colonic mucosa, and several colon cancer cell lines (Fig. 1E).